This invention was made with government support under 1R01DK41921-03, 1R01DK43859-01, and 1P01DK44239-10A1 by the National Institutes of Health. The government has certain rights in the invention.
1. Field of the Invention
This invention relates to adrenocorticotropic hormone receptors from mammalian species and the genes corresponding to such receptors. Specifically, the invention relates to the isolation, cloning and sequencing of a human adrenocorticotropic hormone receptor gene. The invention also relates to the isolate, cloning and sequencing of a bovine adrenocorticotropic hormone receptor gene. The invention relates to the construction of eukaryotic recombinant expression constructs capable of expressing these adrenocorticotropic hormone receptors in cultures of transformed eukaryotic cells, and the production of the adrenocorticotropic hormone receptor in such cultures. The invention relates to the use of such cultures of transformed eukaryotic cells to produce homogeneous compositions of such adrenocorticotropic hormone receptors. The invention also provides culture of such cells producing adrencorticotripic hormone receptor for the characterization of novel and useful drugs.
2. Background of the Invention
The proopiomelanocortin (POMC) gene product is processed to produce a large number of biologically active peptides. Two of these peptides, alpha-melanoctye stimulating hormone (xcex1MSH), and adrencorticotripic hormone (ACTH) have well understood roles in control of melanocyte and adrenocortical function, respectively. Both of the hormones, however, are found in a variety of forms with unknown functions. The melanocortin peptides also have a diverse array of biological activities in other tissues, including the brain, and immune system and bind to specific receptors there with a distinct pharmacology [see, Hanneman et al., in Peptide Hormone as Prohormones, G. Martinez, ed. Ellis Horwood Ltd.: Chichester, UK pp. 53-82; Dewied and Jolles, 1982, Physiol. Rev. 62: 976-1059 for reviews].
A complete understanding of these peptides and their diverse biological activities requires the isolation and characterization of their corresponding receptors. Some biochemical studies have been reported in the prior art.
Oelofsen and Ramachandran, 1983, Arch. Biochem. Biophys. 225: 414-421 disclose receptor binding studies on ACTH receptors on rat adipocytes.
Mertz and Catt, 1991, Proc. Natl. Acad. Sci. USA 88: 8525-8529 disclose functional expression of ACTH receptors in Xenopus laevis oocytes following injection of total cellular RNA from adrenal tissue.
Moore et al., 1991, Endocrinology 34: 107-114 relates to Allgrove syndrome, an autosomal recessive syndrome characterized by ACTH insensitivity.
The present invention comprises a human adrencorticotropic hormone receptor gene, the nucleotide sequence of this gene and the deduced amino acid sequence of its cognate protein, a homogeneous composition of the adrenocorticotropic hormone receptor, nucleic acid hybridization probes and a method for determining the tissue distribution of expression of the gene, a recombinant expression construct capable of expression the gene in cultures of transformed eukaryotic cells, and such cultures of transformed eukaryotic cells useful in the characterization of novel and useful drugs. The present invention also comprises the bovine adrenocorticotropic hormone receptor gene.
The present invention relates to the cloning, expression and functional characterization of mammalian adrenocorticotropic hormone receptor ACTH-R genes. The invention comprises the nucleotide sequence of these genes encoding the mammalian ACTH-RS and the deduced amino acid sequences of the cognate proteins, as well as tissue distribution patterns of expression of these genes.
In particular, the present invention is directed toward the isolation, characterization and pharmacological use of the human ACTHR, the gene corresponding to this receptor, a nucleic acid hybridization probe comprising DNA sequences of the human ACTH-R, a recombinant eukaryotic expression construct capable of expressing the human ACTH-R in cultures of transformed eukaryotic cells and such cultures of transformed eukaryotic cells that synthesize the human ACTH-R, a homogeneous composition of the human ACTHR, and antibodies against and epitopes of the human ACTH-R.
The present invention is also directed toward the isolation, characterization and pharmacological use of the bovine ACTH-R, the gene corresponding to this receptor, a nucleic acid hybridization probe comprising DNA sequences of the bovine ACTH-R, a recombinant eukaryotic expression construct capable of expressing the bovine ACTH-R in cultures of transformed eukaryotic cells and such cultures of transformed eukaryotic cells that synthesize the bovine ACTH-R, a homogeneous composition of the bovine ACTHR, and antibodies against and epitopes of the bovine ACTH-R.
It is an object of the invention to provide a nucleotide sequence encoding a mammalian ACTH-R. In a preferred embodiment of the invention, the nucleotide sequence encodes the human ACTH-R. In another preferred embodiment, the nucleotide sequence encodes the bovine ACTH-R.
The present invention includes a nucleotide sequence encoding a human ACTH-R receptor derived from a DNA molecule isolated from a human genomic library (SEQ ID NO:5). In this embodiment of the invention, the nucleotide sequence includes 2028 nucleotides of the human ACTH-R gene comprising 893 nucleotides of coding sequence, 696 nucleotides 5xe2x80x2 untranslated sequence and 439 nucleotides of 3xe2x80x2 untranslated sequence.
The present invention also includes a nucleotide sequence encoding a bovine ACTH-R derived from a cDNA molecule isolated for a cDNA library constructed with bovine RNA (SEQ ID NO:3). In this embodiment of the invention, the nucleotide sequence includes 1106 nucleotides of the bovine ACTHR gene comprising 893 nucleotides of coding sequence, 133 nucleotides of 5xe2x80x2 untranslated sequence and 82 nucleotides of 3xe2x80x2 untranslated sequence.
The invention includes nucleotide sequences of mammalian ACTH-R, most preferably bovine and human ACTH-R (SEQ ID NOs:3and5), and includes allelic variations of these nucleotide sequences and the corresponding ACTH-R molecule, either naturally occurring or the product of in vitro chemical or genetic modification, each such variant having essentially the same nucleotide sequence as the nucleotide sequence of the corresponding ACTH-R disclosed herein, wherein the resulting ACTH-R molecule has substantially the same biological properties as the ACTH-R molecule corresponding to the nucleotide sequence described herein. The term xe2x80x9csubstantially homologous toxe2x80x9d as used in this invention encompasses such allelic variability as described in this paragraph.
The invention also includes a predicted amino acid sequence for the bovine (SEQ ID NO:4) and human (SEQ ID NO:6) ACTH-R deduced from the nucleotide sequence comprising the complete coding sequence of the bovine (SEQ ID NO:3) and human (SEQ ID NO:5) ACTH-R gene as described herein.
In another aspect, the invention comprises a homogeneous composition of 34 kilodalton bovine ACTH-R or derivative thereof, wherein the amino acid sequence of the ACTHR or derivative thereof comprises a sequence shown in FIG. 3 (SEQ ID NO:4).
In another aspect, the invention comprises a homogeneous composition of a 34 kilodalton human ACTH-R or derivative thereof, wherein the amino acid sequence of the ACTHR or derivative thereof comprises a sequence shown in FIG. 3 (SEQ ID NO:6).
This invention provides both nucleotide and amino acid probes derived from these sequences. The invention includes probes isolated from either cDNA or genomic DNA clones, as well as probes made synthetically with the sequence information derived therefrom. The invention specifically includes but is not limited to olignonucleotide, nick-translated, random primed, or in vitro amplified probes made using cDNA or genomic clone embodying the invention, and oligonucleotide and other synthetic probes synthesized chemically using the nucleotide sequence information of cDNA or genomic clone embodiments of the invention.
It is a further object of this invention to provide sequences of mammalian ACTH-R, preferably the bovine or human ACTH-RS, for use as nucleic acid hybridization probes to determine the pattern, amount and extend of expression of this receptor in various tissues of mammals, including humans. It is also an object of the present invention to provide nucleic acid hybridization probes derived from the sequences of the bovine or human ACTH-RS used for the detection and diagnosis of genetic diseases. It is an object of this invention to provide nucleic acid hybridization probes derived from the DNA sequences of the bovine or human ACTH-R to be used for the detection of novel related receptor genes.
The present invention also includes synthetic peptides mades using the nucleotide sequence information comprising cDNA or genomic clone embodiments of the invention. The invention includes either naturally occurring or synthetic peptides which may be used as antigens for the production of ACTH-R specific antibodies, or used for competitors of the ACTHR molecule for drug binding, or to be used for the production of inhibitors of the binding of agonists or antagonists or analogues thereof to ACTH-R molecule.
The present invention also provides antibodies against and epitopes of mammalian ACTHRs, preferably bovine or human ACTH-R proteins. It is an object of the present invention to provide antibodies that is immunological reactive to a mammalian ACTH-R protein. It is a particular object of the invention to provide a monoclonal antibodies to mammalian ACTH-R protein, most preferably bovine or human ACTH-R protein.
It is also an object of the present invention to provide a hybridoma cell line that produces such an antibody. It is a particular object of the invention to provide a hybridoma cell line that is the result of fusion between a non-immunoglobulin producing-bovine-myeloma cell line and spleen cells derived from an animal immunized with a human cell line which expresses ACTH-R antigen. The present invention also provides a hybridoma cell line that produces such an antibody, and that can be injected into a living animal to provide an ascites fluid from the bovine that is comprised of such an antibody.
The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody that is immunological reactive to a mammalian ACTH-R, preferably a bovine or human ACTHRR, and in a pharmaceutically acceptable carrier.
It is a further object of the present invention to provide an epitope of a mammalian ACTH-R protein wherein the epitope is immunological reactive to an antibody specific for the mammalian ACTHR. In preferred embodiments, the epitope is derived from bovine or human ACTH-R protein.
It is another object of the invention to provide a chimeric antibody that is immunologically reactive to a mammalian ACTH-R protein. In a preferred embodiment, the chimeric antibody is a monoclonal antibody. In a preferred embodiment, the ACTH-R is a bovine or human ACTH-R.
The present invention provides a recombinant expression construct comprising the nucleotide sequence of a mammalian ACTH-R, preferably the bovine or human ACTH-R and sequences sufficient to direct the synthesis of bovine or human ACTH-R in cultures of transformed eukaryotic cells. In a preferred embodiment, the recombinant expression construct is comprised of plasmid sequences derived from the plasmid pcDNAI/neo and cDNA or genomic DNA of bovine or human ACTH-R gene. This invention includes a recombinant expression construct comprising essentially the nucleotide sequences of genomic or cDNA clones of bovine or human ACTH-R in an embodiment that provides for their expression in cultures of transformed eukaryotic cells.
It is also an object of this invention to provide cultures of transformed eukaryotic cells that have been transformed with such a recombinant expression construct and that synthesize mammalian, preferably bovine or human, ACTH-R protein. In a preferred embodiment, the invention provides human 293 cells that synthesize bovine ACTH-R. In an additional preferred embodiment, the invention provides human 292 cells that synthesize human ACTH-R protein.
The present invention also includes protein preparations of mammalian, preferably bovine or human ACTH-R, and preparations of membranes containing mammalian ACTH-R, derived from cultures of transformed eukaryotic cells. In a preferred embodiment, cell membranes containing bovine ACTH-R protein are isolated from 293 cell cultures transformed with a recombinant expression construct that directs the synthesis of bovine ACTH-R. In another preferred embodiment, cell membranes containing human ACTH-R protein are isolated from 293 cell cultures transformed with a recombinant expression construct that directs the synthesis of human ACTH-R.
It is also an object this invention to provide mammalian, preferably bovine or human ACTH-R for use in the in vitro screening of novel-adenosine-agonist and antagonist compounds. In a preferred embodiment, membrane preparations containing the bovine ACTH-R, derived from cultures of transformed eukaryotic cells, are used to determine the drug dissociation properties of various novel agonist and antagonist compounds in vitro. In another preferred embodiment, membrane preparations containing the human ACTH-R, derived from cultures of transformed eukaryotic cells, are used to determine the drug dissociation properties of various novel-adenosine agonist and antagonist compounds in vitro. These properties are then used to characterize such novel compounds by comparison to the binding properties of known bovine or human ACTH-R agonists and antagonists.
The present invention is also useful for the in vivo detection of analogues of agonists or antagonists of ACTH-R, known or unknown, either naturally occurring or as the embodiments of a drug.
It is an object of the present invention to provide a method for the quantitative detection of agonists or antagonists, or analogues thereof, ACTH-R, known or unknown, either naturally occurring or as the embodiments of a drug. It is an additional object of the invention to provide a method to detect such agonists, antagonists, or analogues thereof in blood, saliva, semen, cerebrospinal fluid, plasma, lymph, or any other bodily fluid.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.